Journal of Agricultural and Food Chemistry 0 Copyright 1972 b y the American Chemical Society
Volume 20,Number I
JanuarylFebruary 1972
Comparative Metabolism of DDT, Methylchlor, and Ethoxychlor in Mouse, Insects, and in a Model Ecosystem l n d e r P. Kapoor, R o b e r t L. Metcalf,* Asha S. Hirwe, Po-Yung Lu, Joel R. Coats, and Robert F. Nystrom
Ethoxychlor [2,2-bis(p-ethoxyphenyl)-l,l,l-trichloroethane] and methylchlor [2,2-bis(p-methylphenyl)1,l ,I-trichloroethane] were evaluated for metabolic pathways in mice and insects and for biodegradability in a model ecosystem. Ethoxychlor, like methoxychlor, was metabolized by 0 dealkylation to form 2-(p-hydroxyphenyl)-2-(p-ethoxyphenyl)-l,l,1-trichloroethane and subsequently to 2,2-bis(phydroxypheny1)-1,l ,I-trichloroethane. In the model ecosystem ethoxychlor was found in fish at the top of the food chain at 1500 times the amount in water, together with larger amounts of polar and dealkyla-
n the search for biodegradable analogs of DDT, the metabolism of methoxychlor and methiochlor in mice, insects, and a model ecosystem was investigated by Kapoor et al. (1970). In further study of the ecological biochemistry of DDT analogs with biodegradable substituents in the para positions of the aryl rings, ethoxychlor or 2,2-bis(pethoxypheny1)-1,1 ,1-trichloroethane and methylchlor or 2,2bis(pmethylpheny1)-l,l,l-trichloroethane have now been investigated. Ethoxychlor was first synthesized by Fritsch and Feldman (1899). It is substantially more insecticidal than methoxychlor (Metcalf and Fukuto, 1968) but is also more toxic to mammals with an oral LDjo to the mouse of 300 mg/kg (Von Oettingen and Sharpless, 1946). Methylchlor was first made by Fisher (1874). It was found to be substantially toxic to insects (Metcalf and Fukuto, 1968) and nontoxic to the mouse, oral L D ~ o> 1000 mg/kg (Von
I
Departments of Entomology and Zoology, University of Illinois, Urbana, Illinois 61801.
tion products. Methylchlor was metabolized by oxidation of the arylmethyl groups t o form 2-(phydroxymethylpheny1)-2-(p-methylpheny1)- l,l,l-trichloroethane and by subsequent oxidation to 2,2bis(p-carboxypheny1)-1,1,1 -trichloroethane. In the model ecosystem, methylchlor was found in fish at 1400 times the amount in water together with larger amounts of polar metabolites. These two DDT analogs are much more biodegradable than DDT, which was concentrated in fish at 85,000 times the amount in water.
Oettingen and Sharpless, 1946). Ariens (1966) suggested, without evidence, that methylchlor should be substantially biodegradable through microsomal side chain oxidation to hydroxymethyl and benzoic acid moieties. Peterson and Robison (1964) and Jensen et al. (1957) have reported the qualitative metabolism of DDT in mammals, and it is surprising that even now there is little data available on its metabolic pattern in the mouse. Due to this paucity of information, the problem has been restudied and information is presented on the metabolism of DDT, methylchlor, and ethoxychlor. MATERIALS A N D METHODS
Radio-Labelled Compounds. SH-Ring substituted ethoxychlor was synthesized by the method of Hilton and O'Brien (1964), and purified by column chromatography on silica gel by elution with 2% diethyl ether in petroleum ether (boiling range 60-68" C). The product had a radio purity of 99.9f evaluated by thin-layer chromatography (tlc), J. AGR. FOOD CHEM., VOL. 20, NO. 1, 1972 1
METCALF
et al.
Table I. Properties of Ethoxychlor and Its Model Metabolites Thin-layer Detectionb chromatography uv D-Z mp, "C HDEl HDEz Compound CZH,OC~H~HCCC~,C,H~O~~H~ 105 0.49 0.71 None Steel gray 0.55 0.71 None Pink CZH,OC,H~CCC~ZC~H~OCH~ 107 GH5OCgH4HCCCIgC6H4OCH2CH20H 117 0.40 None Steel gray CZH~OC~H~HCCC~~C~H~OH 65-7 0.49 Light yellow Black 194.0 0.27 Yellow Black HOC~H~HCCC~~CE,H~OH 212.0 0.30 Yellow Pink HOC~H~CCC~ZCGH~OH Yellow None HOC6H4COCsHaOH 213-215.0 0.18 Color a The development systems: HDEl = hexane-dioxane-diethyl ether (80:20:10) ; HDEz = hexane-dioxane-diethyl ether (90:60:10). zinc chloride (in acetone), heating at 110" C for detection: uv = exposure to uv light for 5-10 min; D-Z = spraying with 0.5 % diphenylamine 10 min and exposure to uv light for 5 min.
+
Table 11. Properties of Methylchlor and Its Model Metabolites Thin-layer chromatography (Rf)" Compound mp, "C P BDA 0.28 0.71 CH~C~H~HCCC~ZC~H~CH~ 83 0.38 0.71 CH~C~~H~CCC~ZC~H~CH~ 82 0.53 C H ~ C ~ H ~ H C C C ~ ~ C ~ H ~ C H Z O H 74 0.48 CH~C~H~HCCC~~CGH~COOH 107 0.30 H O H Z C C ~ H ~ H C C C ~ ~ C ~ H ~ C H Z OGummy H solid 0.33 HOOCCgHpHCCCIaC6HaCOOH 274-6
Detectionb D-Z Black Pink Gray Gray None None
Color detection: TIC development systems: P = petroleum ether (60-68," C); BDA = benzene-90 to dioxane-30 to acetic acid (glacialbl. D-Z = spraying with 0.5 % diphenylamine zinc chloride (in acetone), heating at 110" C for 10 mm and exposure to uv light for 5 min.
+
using solvent HDE (Table I) with a specific activity of 4.44 mCi/mmol. 14C-Methyl-labelled methylchlor [2,2-bis(p-methylphenyl)1,l,l-trichloroethane] was synthesized in vacuum line from 3 mg of 14C-toluene, diluted to 20 mg with cold toluene, and condensed with 20 mg of chloral in 10 volumes of concentrated HzS04in 6 0 z yield. The product was purified by column chromatography on silica gel with petroleum ether (60-80" C) as the eluent, and had a radiochemical purity of 99.9+ with a specific activity of 1.30 mCi/mmol. l*C-Ring-labelled D D T was supplied by the World Health Organization, Geneva, Switzerland, with a specific activity of 5.48 mCi/mmol. It was purified by column chromatography to 99.9+ purity. Radioassay. Tritium-labelled compounds in biological materials were assayed by oxygen flask combustion. 14C-Labelled compounds likewise were also combusted by a procedure essentially identical to tritium combustion. For COz absorption, 20 ml of N cocktail (135 ml of phenethylamine, 135 ml of methanol, 730 ml of toluene, 5 g of PPO, and 100 mg of POPOP) was injected into the flask after combustion. The flask was held over ice cold water for 15 min and periodically shaken. A 15-mlaliquot was then counted on a Packard 2002 liquid scintillation counter. Other radioassay methods were those of Kapoor et al. (1970). Chromatographic techniques were carried out on 0.25 mm plates of silica gel with a fluorescent indicator (E. Merck) using the solvent systems given in Tables I and 11. Chromogenic techniques for identification of individual metabolites are given by Kapoor et a/. (1970). Model Metabolites. The previous investigation of the metabolism of methoxychlor (Kapoor et al., 1970) which showed biological degradation to mono- and dihydroxyphenols produced by 0 dealkylation suggested that ethoxychlor might be degraded through a similar metabolic pathway. Therefore, the following model metabolites were prepared. Ethoxychlor or 2,2-bis(p-ethoxyphenyl)-l,l,l-trichloroethane (I), mp 105" C (Fritsch and Feldman, 1899; mp 105" C) was synthesized by condensation of ethoxybenzene (phenetole) with chloral in the presence of A1C13.
z
z
2 J. AGR. FOOD CHEM., VOL. 20, NO. 1, 1972
The 2,2-bis(pethoxyphenyl)-l,l-dichloroethylene (11), mp 107" C (Stephenson and Waters, 1946; mp 109" C) was obtained from I by alkaline dehydrochlorination in ethanol. 2-p-Ethoxyphenyl-2- p - @-hydroxyethoxyphenyl) - 1,1,1-trichloroethane (111) was obtained by reacting 2.0 g of IV with 1.0 g of 2-bromoethanol in 50 ml of dry acetone containing 2.0 g of K2C03. The mixture was refluxed for 14 hr, filtered, and acetone removed under reduced pressure. The residue was taken up in ether, washed twice with 5 KOH and then water. The product was purified on a silica gel column by eluting with increasing concentrations of diethyl ether in petroleum ether (60-68 O C). The product was recrystallized from cyclohexane to mp 107" C and identified by nmr spectrometry, showing a - H at T 5.07, -CH3 at T 8.63, -CH2 at T 6.07, and -OH at 7 7.88. 2-p-Ethoxyphenyl-2-p-hydroxyphenyl- 1,1,1-trichloroethane (IV), mp 65-67" C, was prepared by the method of Kapoor et al. (1970) for the methoxy analog. Nmr spectrometry showed a-H at T 5.03, -CH3 at T 8.62, and -CH2 at T 5.94. Methylchlor or 2,2-bis(p-methylphenyl)-l,l,l-trichloroethane (VIII), mp 87-88' C (Fisher, 1874; mp 89" C) was obtained by condensation of 18.4 g of toluene with 14.8 g of chloral by the dropwise addition of 250 ml of concentrated HzS04. The product was crystallized from ethanol. 2,2-Bis(p-methylphenyl)-l,l-dichloroethylene (IX), mp 82 " C (Stephenson and Waters, 1946; mp 87" C) was prepared by alkaline dehydrochlorination in ethanol. 2-p-Methylphenyl-2-p-hydroxymethylphenyl- 1,1,1-trichloroethane (X) was made from 2-p-methylphenyl-2-p-bromomethylphenyl-l,l,l-trichloroethane (from the bromination of VIII, identified by nmr, -CHz at T 5.57, -CH3 at T 7.88, and a-H at T 5.01) by base hydrolysis and purified by column chromatography on silica gel with increasing concentrations of diethyl ether in petroleum ether (60-68" C) as the eluent. The product was crystallized from petroleum ether, mp 74" C. Nmr spectrometry showed a-H at T 4.92, -CH3 protons at T 7.65, -CHz at T 5.28, and -OH at T 8.32, confirmed by its disappearance in the presence of D 2 0 . 2,2-Bis(p-hydroxymethylphenyl)-l,l,l-trichloroethane(XI)
z
COMPARATIVE METABOLISM OF DDT, METHYLCHLOR, AND ETHOXYCHLOR
was prepared from 2,2-bis(p-bromomethylphenyl)-l,1,1 -trichloroethane (Stephenson and Waters, 1946) by alkaline hydrolysis. The resultant product was a gum, even after purification by column chromatography. The product gave a single spot o n tlc. Nmr spectrum confirmed the structure by presence of -OH at 7 7.4 which disappeared in the presence of D20. 2-p-Methylphenyl-2-p-carboxyphenyl- 1,1,1-trichloroethane (XII) was made by oxidation of VI11 with CrOs in glacial acetic acid and crystallized from dilute ethanol to mp 107" C and identified by infrared spectrometry (C=O at 1680 cm-1) and nmr data which showed a-H at 7 4.88, -CH8 at 7 7.72, and -OH at 7 1.34. Fisher (1874) gives 174" C, which must be a n error. 2,2-Bis(p-carboxyphenyl)-l,l,l-trichloroethane(XIII), mp 274-276" C, was obtained from VI11 (Haskelberg and Lavie, 1949; mp 271-272" C). Ir showed strong absorption at 1600 cm-'.
100
P
$
METHOXYCHLOR
0
METHIOCHLOR
0
ETHOXYCHLOR
'(
n IL
TOXICOLOGICAL METHODS
Metabolic studies in male Swiss mice were carried out by oral administration of I4C-DDT, "C-methylchlor, and 3H-ethoxychlor at 50 mg/kg in olive oil. Procedures for mice, houseflies, and salt marsh caterpillars have already been described (Kapoor et a/., 1970). Microsomal Oxidations. Ethoxychlor and methylchlor were incubated with mouse liver homogenate. After perfusion with 1.15% KCl, mouse liver was homogenized and suspended in 1.15% KC1 ( 1 0 z w/v). The homogenate was centrifuged a t 15,000 X g for 30 min and the incubation mixture, consisting of 32 ml of supernatant, 1 ml of NADPH (0.1 1 ml of nicotinamide (5.2 X 10-4 M), 2.6 ml of phosphate buffer (0.2 M , pH 7.8) and 20 pl of substrate in ethanol was incubated for 1 hr at 37" C, acidified to pH 2.0, (0.1 heated on a steam bath for 1 hr, extracted with Et20, and processed for identifying and determining the metabolites. Model Ecosystem. Detailed methodology has already been reported (Kapoor et al., 1970; Metcalf et al.,1971b).
z), z),
RESULTS A N D DISCUSSION
Studies of the metabolic fate of radio-labelled ethoxychlor and methylchlor in RsP female houseflies are shown in Table 111. The percent recovery for ethoxychlor was poor and the data are the average of three different experiments. Analysis of the results shows that ethoxychlor is 0 dealkylated t o monoand bis-phenolic products, but unlike methoxychlor, dehydrochlorination does play an important role. Since 0 deethylation is an inefficient process compared to 0 demethylation (Hansen et al., 1971), the organism must utilize the alternative dehydrochlorination as a survival mechanism. Methylchlor is oxidized t o benzyl alcohol and benzoic acid analogs. Metabolism by Salt Marsh Caterpillar. Substantially more radioactivity fed the organism was recovered in the feces in case of ethoxychlor (99.0 %) than methylchlor (93.6 %). Whereas dehydrochlorination was the major metabolic pathway for ethoxychlor metabolism, methylchlor was also oxidized t o benzoic acid analogs and conjugates were formed. The qualitative and quantitative distribution of metabolites is shown in Table IV. Rates of Excretion in the Mouse. Comparative rates of elimination of radio-labelled DDT, ethoxychlor, and methylchlor, along with data for methoxychlor and methiochlor (Kapoor et al., 1970), are shown in Figure 1. The rate of elimination of DDT was determined by the more efficient 0 2 flask combustion technique at 50 mg/kg dosage, equivalent t o the dose used for other analogs. DDT was eliminated
1
1
2
3
4
DAYS
5 AFTER
6
7
8
9
1011
TREATMENT
Figure 1. Accumulative rates of elimination of 'IC-DDT, 3Hethoxychlor, 14C-methylchlor,and 3H-methoxychlorand methiochlor in urine and feces of mouse following oral administration. Data for methoxychlor and methiochlor from Kapoor et a/. (1970) Table 111. Metabolism of Ethoxychlor and Methylchlor in RSP Housefly Percent total radioactivity Homogenate Excreta Ethoxychlor treatment (1.8% wash, 35.8% excreta, 11.0% homogenate = 48.6% recovery) 53.5 14.6 Ethoxychlor GH~OCGH,CCCI~CGHCOC~H~ 21.2 13.4 C2HjOC6H4HCCCI,C6H,OCHtCH20H 12.2 1.3 14.0 C2HiOCeH,HCCCIxCfiH,OH HOC~HICCCI~C~H~OH HOC~HICOC~HIOH Conjugates
4.6
18.b
1.7
11.8 5.9
8.6
7.5
Methylchlor treatment (3.0% wash, 61.0z excreta, 5 % homogenate = 69 recovery) Methylchlor 40.2 18.6 Unknown"
12.4 16.5
CH~C~H~HCCC~~C~HICH~OH HOCHaCsH4HCCC13C6H4CH20H
41.6
HOOCC~H~HCCCIQC~H~COOH Conjugates
18.2
a
12.6 15.1 24.8
Unknown Rr 0.64 (benzene/dioxaiie:'acetic acid 90 :30: 1).
Table IV. Metabolism of Ethoxychlor and Methylchlor in Salt Marsh Caterpillar (Esrigmine acrea) Percent total radioactivity Homogenate Excreta Ethoxychlor treatment (99.0% excreta, 1.0% homogenate) Ethoxychlor 81.5 92.1 C2H5OC6H aCCC12CsH4OC2H5 17 9 6.9 C2H jOC 6H 4HCCC13CsHIOH Traces Traces Trarpc
z
Methylchlor treatment (93.6 excreta, 6.4% homogenate) Methylchlor 84.5 95.5 C H ~ C ~ H ~ C C C I ~ C ~ H I C H ~ 7.4 CH~CGH~HCCCI~C~H~CH~OH Traces HOOCC~H~HCCCIQC~H~COOH 1.4 8.1 2.1 Conjugates J. AGR. FOOD CHEM., VOL. 20, NO. 1, 1972 3
METCALF
Table V. Metabolism of Ethoxychlor and Methylchlor by Mouse Liver Homogenate Percent total radioactivity Ethoxychlor treatment
Ethoxychlor CzHsOCgH4HCCClgCgHaOH Conjugates
72.5 9.1 12.4
Methylchlor treatment Methylchlor unknown I (Rf 0.64)" CH,CsH,HCCCl,C6H,CHzOH CHaC6H4HCCC13CsHaCOOH HOOCC6H4HCCC13CgHaCOOH Unknown I1 (Rf0.32)" HOHZCC~H~HCCCI~CJI,CH~OH Conjugates a
19.3 2.1 30.4 5.5 6.2 7.6 19.8 1.6
Solvent system : benzene/dioxane/acetic acid 90 : 30 : 1.
Table VI.
Feces Polar
Ethoxychlor treatment (feces 62.3 %, urine 37.7 %) 11.5 53.1 1.0 1.2 1.7 9.7 2.1 31.3
88.5
44.5 15.1 1.9 46.0 35.1
3.0 1.4 2.8 92.2
55.5 1.5 18.0 12.3 26.4 6.4 12.1 23.3
Methylchlor treatment (feces 88.9%, urine 11.1 %) 9.1 74.9
Methylchlor CH~C~H~HCCC~~C~HICHZOH CH3C6HaHCCC13C6H4COOH Unknown I (Rf0.44)" Unknown I1 (Rf 0.40)" HOOCC~HIHCCCI~C~H~COOH Unknown 111 (Ri 0.35)"
90.9
4.9 72.6 8.2 9.5
28.0 16.7 19.9
HOHzCCsHiHCCClgCgH4CHgOH Unknown IV (Rf 0.18p
Conjugates
25.1
35.4
9.7
95.2 5.3
21.8 2.4 63.9 1.2 4.5
DDT treatment (feces 73.02%, urine 26.98%) DDE DDT DDMS DDD DBP Kelthane Unknown 1 (Rf0.64)b DDA Unknown I1 (Rf 0.47)b Unknown 111 (Rf 0.40)b Unknown IV (Rf 0.25)b Unknown V (Rf O.ll)b Base (conjugates) Solvent system :
43.3 41.5 29.1 1.4 1.7 2.4 1.9 3.3 14.9 1.2 1.3 1.5
benzene/dioxane/acetic acid 90:30: 1.
4 J. AGR. FOOD CHEM., VOL. 20, NO. 1, 1972
01.
slowly (7.4% in the first 24 hr), while methylchlor (43.7%) and ethoxychlor (99.0 %) were rapidly eliminated. However, over a period of 11 days, 90.8% DDT had been eliminated compared to 73.7% for methylchlor and 77.5% for ethoxychlor, thus indicating that although initially DDT has a much slower rate of elimination, it reaches the level of the other analogs within 6 days. The degree of polarity of the excretory metabolites is indicated by the urine/feces ratio, which is 0.37 for DDT, 0.69 for ethoxychlor, and 0.12 for methylchlor. The ratio for ethoxychlor differs substantially from its methoxy analog (0.13) and may be indicative of its higher toxicity. Metabolism by Mouse Liver Homogenate. As shown in Table V, 72.5% of ethoxychlor was recovered intact after incubation with the homogenate along with 9.1 % of the mono-phenolic product 2-(p-hydroxyphenyl)-2-(p-ethoxyphenyl)-l,l,l-trichloroethaneand 12.4% conjugates. In comparison, 80.7 % of methylchlor was metabolized, the major components being the monoalcohol (30.4 %) or 2-(p-methylphenyl)-2-(p-hydroxyrnethylphenyl)-l,1,1 -trichloroethane and di-alcohol (19.8 %) or 2,2-bis(p-hydroxymethylphenyl)-l,l,l-
Metabolism of DDT, Ethoxychlor, and Methylchlor in Mouse Percent total radioactivity __ Urine Hexane Polar Hexane
Ethoxychlor CZHEOC~H~CCC~ZC~H~OC~HE
et
* Solvent system:
56.7 5.3 2.6
47.8 2.1 28.0
52.2
1.0
61.3
13.0
0.8 4.5 30.6 2.8 3.5 15.7 22.7 10.5
Trace
Trace
3.2
8.7
benzene'dioxane'acetic acid 110:30: 1 , 5 .
28,2 10.1
24.0 21.6
COMPARATIVE METABOLISM OP
om, METHYLCHLOR, AND ETHOXYCHLOR
trichloroethane along with mono- and his-benzoic acid analogs, two unknowns and conjugates. These data suggest that the mammalian liver microsomes are more active in side chain oxidation than in 0 deethylation. Recent studies in our laboratory (Hansen et al., 1971) on comparative metabolism of M methoxychlor and ethoxychlor hy mouse liver microsomes have shown that methoxychlor is metabolized 2.5 times faster than ethoxychlor, explaining the relatively higher toxicity of the latter. DDT, Ethoxychlor, and Methylchlor Metabolism in the Mouse. The metabolism of DDT has been reported previously hy Jensen et a/. (1957) and Peterson and Rohison (1964), hut even today no clear picture is available for its complete qualitative and quantitative metabolic pattern, which is given in Table VI and Figure 2. Over a period of 48 hr 73.0x of the recovered radioactivity was in the feces and the remaining 27.0x in urine. After the analysis it was shown that DDE (41.5%) was the major component in the nonpolar (hexane) fraction of the urine, along with DDT (29.1 %) and DDA (14.9 %), whereas the polar fraction comprised 30.6% DDA and a major portion of more polar unknowns. In the fecal hexanefraction, DDD (61.3z) was the mnior nrnrliirt ._ll_.l^_..nlonv with DDT (28.0%), D D E (2.1 %), and an unknown (8.7%), where:as DDA (28.2%), and D D D (13.0x) together with unknown:3 constituted the polar fraction. It is evident that D D T is hic,logically degradable, hut it is the degradation products, especially DDE, that turn out to he environmental hazards. As shown in Table VI, ethoxychlor like methoxychlor is 0 dealkylated to the phen olic products 2-p-(ethoxyphenyl)-2-(phydroxyphenyl)-1,l ,l-tnichloroethane and 2,2-his(p-hydroxy. L e I, 1 1 1 " ~ " ~ ~ a n eThe . other major identified (p-ethoxyphenyl)-2-(ydroxyethoxy-
y.....~.,-.,.,.-..,~~..
Figure 2. mwioaurograpn 01 mm-layer piare snowing metabolic pattern of W2.DDT by the mouse
phenyl)-l,l,l -tricbloroethane. Conjugates and polar material constituted a h o s t all of the urine polar fraction and considerable amountsi in the other fractions. Methylchlor is also subject to oxidative degradation to alcoholic and carboxylic acid components. The urine fractions were comprised of parent material and Z,Z-bis(pcarboxypheny1)-1.1.1-trichloroethane along with unknowns ,, and polar conjugiates. The dialcoholic 1:AC#duct2,2-bis(p-hy droxymethylphen~yl)-l,l,I-trichloroethane aind the di-acid 2,Zbis(p-carhoxyphenyl)-l,l,l -trichloroethaoe 'Formed the major - LL. r. ..- _L. . ...7.^i^ 2 : .. metabolic produua : 11, LIE ICCTS. I I K reSurs illr g w n ~ 111 Table V. Metabolism in Model Ecosystem. The distribution of 3H-ethoxychlor and '4C-methylchlor and their metabolites is presented in Table VII. Ethoxychlor was concentrated .L.
hlor and Their Metabolites in a Model Ecosystem
Distribution of
woni
Jgae) Ethoxychlor Total aH Unknown I
0.0904
2.014
O.OCQ6
1,526 0.039 0.355 0.041 0.053
thylchlor 0.2159
5,525
0.0006
5.168
0.0233
0.124
0.0343 0.0167 0.0032 0.1149
0.233
METCALP et
Figure 3. Radioautograph of thin-layer plate showing metabolic pattern of degradation of 14C-methylchlorby elements of a model ecosystem. 1. CH3CoH4HCCCl3CsH4CHa;2. CHsCsH4HCCC1aCaHEH20H; 3. CH3CsHbHCCCLCBH6C0OH; 4. HOOCC~H~HCCC~~C~HICOOH
al.
Figure 5. Pathways of methylchlor metabolism in mouse, housefly, and salt marsh caterpillar SUMMARY AND CONCLUSIONS
The pathways of metabolism for ethoxychlor and methylchlor in insects, mice, and in the model ecosystem (Figures 4 and 5 ) demonstrate that these compounds, like methoxychlor and methiochlor previously investigated (Kapoor et ai., 1970) are persistent, biodegradable analogs of DDT. It is evident that the CHr, CH&, C,H,O-, and C H & moieties can serve as degradative handles in the D D T type molecule and that the incorporation of any one of these can markedly accelerate the biodegradability of the molecule. This information has been used to synthesize a number of new asymmetrical D D T analogs, highly toxic to insects and of greatly reduced toxicity to the mouse. Ethoxychlor is more toxic than DDT to some insects and both ethoxychlor and methylchlor are much effective than D D T to the moderately DDTresistant housefly (Metcalf et a/., 1971a). This can be attributed to a lower rate of dehydrochlorination because of the electron donating C2HsO- and CHr groups (Metcalf et al., 1968).
(M.4
Figure 4. Pathways of ethoxychlor metabolism in mouse, housefly, and salt marsh caterpillar
in the higher positions of the food chain, e.g., in fish, to a level of 1500 times that in the water. This represented only 19.2% of the total activity in the organism, the major constituent being the monophenolic product 2-(p-ethoxyphenyl)2 - ( p - hydroxyphenyl) 1,1,1 - trichloroethane. Snails concentrate large quantities of ethoxychlor (98,000 fold) over that present in water, which is at a much higher level than DDT (35,000 fold) but less than the concentration of methoxychlor of 120,000 fold (Kapoor et a/., 1970). With methylchlor, the parent material was stored in the fish at 1400 times, comparable with other biodegradable analogs investigated. Snails once again concentrated even this compound 120,000 times, indicating a metabolic deficiency of microsomal 0 dealkylation and side chain oxidation. Mono- and bis-alcohols and carboxylic acids were present as metabolites. Radioautographs showing the qualitative metabolic pattern of environmental degradation of methylchlor in the model ecosystem are presented in Figure 3.
-
6 J. AGR. FOOD CHEM., VOL. 20, NO, 1, 1972
LITERATURE CITED
Ariens, E. J., in Progress in Drug Research, E. Jncker, Ed., Brickhaus Verlog, Basel-Stuttgart, X, 1966, p 429. Fisher, O., Ber. Deuts. Chem. Ges. I, 1191 (1874). Fritsch, P., Feldman, F., Justus Ann. Liebigs Chem. 306,72 (1899). Hansen, L. J., Kapoor, 1. P., Metcalf, R. L., unpublished data (1971). ~~.~ , .
Haskelberg, L., Lavie, D., J. Org. Chem. 14,498 (1949). Hilton, B. D., O'Brien, R. D., J. AGR.FOODCHEM. 12, 236 (1964). Jensen, J. S., Cento, C., Dale, W. E., Rothe, C. F., Pearce, G. W., Matteson, A. M., J. AOR.FOOD CHEM. 5,919 (1957). Kapoor, I. P., Metcalf, R. L., Nystrorn, R. F., Sangha, G. K., J. AOR.FOOD CHEM.18(6),1145 (1970). Metcalf, R. L., Fukuto, T. R., BUN. W . H . 0.38,633 (1968). Metcalf, R. L., Kapoor, I. P., Hirwe, A. S., BUN. W . H . 0.44, 363 (1971a). Metcalf,R . L., Sangha, G. K., Kapoor, I. P., Emiron. Sei. Techno/. 5,709 (1971b). Peterson, J. E., Robison, W. H., Toxicol. Appl. Phormocol. 6, 321
.,._ _
1196A) ~.
Stephenson, O., Waters, W. A,, J. Chem. Sac. 339 (1946). Von Oettingen, W. F., Sharpless, N. E,, J. Pharm. Exp. Therap. 88, 400 (1946). Receioed for review Mav 17. 1971. Acceotrd Julv 2. 1971. SUD-
